CN114107465A - Kit for specifically detecting sarcopenia through rs33966734 - Google Patents

Kit for specifically detecting sarcopenia through rs33966734 Download PDF

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CN114107465A
CN114107465A CN202010891138.0A CN202010891138A CN114107465A CN 114107465 A CN114107465 A CN 114107465A CN 202010891138 A CN202010891138 A CN 202010891138A CN 114107465 A CN114107465 A CN 114107465A
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sarcopenia
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张垒
裴育芳
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Suzhou Jinhao Biotechnology Co ltd
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    • C12Q2600/156Polymorphic or mutational markers

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Abstract

The invention discloses a kit for specifically detecting senile sarcopenia by detecting rs 33966734. The kit comprises a specific primer pair and a specific fluorescent probe pair for detecting No. rs33966734 SNP locus, a conventional component for fluorescent quantitative PCR detection and the like. The kit of the invention evaluates the genetic susceptibility of the individual senile sarcopenia by simultaneously detecting the single nucleotide polymorphism locus genotype of the rs33966734 closely related to the genetic susceptibility of the senile sarcopenia.

Description

Kit for specifically detecting sarcopenia through rs33966734
Technical Field
The invention relates to the field of SNP typing detection in the technical field of genetic engineering, in particular to a kit for specifically detecting sarcopenia by detecting rs 33966734.
Background
Sarcopenia, also called Sarcopenia (Sarcopenia), is a chronic metabolic disease of the elderly, which is mainly characterized by the decrease of the quality of skeletal muscle fibers, the decrease of muscle strength, the decrease of muscle endurance and metabolic capability, the increase of connective tissues and fat, and the like with the increase of age. Sarcopenia often causes the mobility of the old to be reduced, daily actions such as walking, sitting, climbing, lifting heavy objects and the like are influenced, and the hand is gradually free from binding the force of chicken. The muscle function is reduced, and the probability of accidental falling injury of the old is increased by 40 percent. Statistically, 50% of the elderly die from accidents due to falls. The risk of inconvenient actions of the sarcopenia old people is 2-5 times that of the old people of the same age, and the life quality and the self-care ability of the old people are seriously influenced.
Sarcopenia not only increases the risk of disability and loss of self-care ability of life, but also promotes the development of diseases such as osteoporosis and arthritis, and is also an important reason for inducing senile diseases such as hypertension, diabetes and hyperlipidemia. Sarcopenia has become one of the leading causes of disability and death worldwide. Sarcopenia occurs in 13-24% of the elderly under age 70, with a prevalence of up to 50% and even higher in those over age 80. Annual economic losses associated with sarcopenia exceed $ 1000 billion.
Numerous studies of candidate genes in the past have shown that many genes are associated with changes in human muscle mass. More than 40% of the genetic variations associated with the human lean body mass index (LMI) can produce differences in traits. With the progress of molecular genetics, it is found that sarcopenia is a polygenic genetic disease, and genetic association research of sarcopenia is carried out in many countries. At least 20 human genes or chromosomal regions have been detected by molecular genetics and reported to be associated with sarcopenia to date. In addition, both genetic and molecular epidemiological evidence suggests that genetic factors are involved in determining susceptibility to increased or lost muscle mass in specific dietary or drug regimes, as well as a high risk of developing other related diseases in sarcopenia patients. At present, many candidate genes exist, but most of the candidate genes are required to be further improved in detection accuracy.
As for the method for detecting sarcopenia genes, the conventional detection method at present includes Restriction Fragment Length Polymorphism (RFLP), which is a method for simply treating the PCR product of a patient by enzyme digestion and then detecting whether there is variation when the variation affects the enzyme cutting site of a certain Restriction enzyme. However, the method has the defects of long time consumption, complex operation, low accuracy and the like. The detection of gene polymorphism sites by PCR combined with DNA sequencing is also useful, but the application of this method in large-scale population screening or detection of multiple sites of multiple genes is limited. Therefore, it is necessary to establish a high-throughput, high-efficiency and low-cost SNP (single nucleotide polymorphism) typing method for sarcopenia susceptibility genes to realize clinical rapid detection or large-scale population screening.
Disclosure of Invention
The invention provides a kit for specifically detecting sarcopenia by detecting rs 33966734.
The invention aims to provide a screening method of SNP molecular markers of sarcopenia susceptibility genes, which is characterized in that a molecular marker locus rs33966734 is found to be positioned on a No. 6 chromosome 41903798 through a genome sequence of a large-scale genetic array sample of a British biological sample library and the muscle content of four limbs measured by a bioelectrical impedance method, and the mutation is A/C which is directly related to the muscle content of a human.
The invention provides an rs33966734 molecular marker amplification primer pair on a 6p21.1 chromosome according to an rs33966734 molecular marker sequence disclosed on NCBI, wherein an upstream primer of the primer pair: TTCCCCATGTGTTGGGAGCT, respectively; a downstream primer: CTCCTCTGCTTAGTGGCCAC, respectively; amplified fragment size: 301 bp. Simultaneously, a pair of detection primer pairs is provided, and genotype-fluorescent probe sequences are as follows: 5 '-FAM-CACTCAGGGAaAGCCTCAGGG-TAMRA-3'; genotype two fluorescent probe sequence: 5 '-VIC-CACTCAGGGAcAGCCTCAGGG-TAMRA-3'.
The rs33966734 SNP detection chip on the chromosome 6p21.1 can be used for detecting the mutation of the A/C of the chromosome 6p21.1 segment individually or in parallel. The detection chip is prepared by adopting a conventional construction method in the field.
The invention provides a kit for detecting sarcopenia genetic susceptibility. The kit comprises:
specific primer pair for detecting rs33966734 SNP polymorphism genotype of SEQ ID NO: 2 and 3; PCR reaction assembly (including Taq enzyme, dNTP mixed solution, MgCl2Solution, reaction buffer, deionized water, etc.).
The main advantages of the invention
The rs33966734 SNP marker identified and obtained from chromosome 6p21.1 can be used for identifying the symptoms of sarcopenia of human beings, and the identification has the advantages of good accuracy and high specificity.
The detection method of the invention has simple steps, the SNP locus detection can be completed by one-step PCR, the amplification of the target sequence containing the SNP locus avoids a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like, thereby greatly improving the detection accuracy and embodying the accurate and simultaneously qualitative and quantitative analysis characteristics.
Detailed Description
EXAMPLE 1 obtaining molecular markers for SNPs
1, sample of
The samples used in the present invention were from a uk biosample bank (UKB) cohort of samples. The cohort was a large prospective study cohort containing 50 million participants between 48-73 years of age from all over the country in the uk. We use a series of strict nanocriterions. Exclusion criteria included inconsistency of self-reported sex with genetic sex, non-ploidy sex chromosomes, too high a heterozygosity, too high a genotype deletion rate, and the like. There were 48.7 million participants, and 450243 participants remained available for analysis after the exclusion of 3.7 million participants, of which 205513 were male and 244730 were female.
2, phenotype and modeling thereof
Body composition including muscle content and fat content was measured using a bioelectrical impedance method. The fat content and the muscle content of the arms and the legs are measured in sequence, and the muscle content of the limbs is obtained by adding the muscle content of the arms and the legs. Male and female are modeled separately. In each sex, the raw limb muscle mass was corrected using the limb fat mass, age square, first ten genetic principal components, the measurement location, and the chip used for genotyping as covariates. The corrected residuals were converted to standard normal distributions according to the inverse quantile method and used for downstream analysis.
3, genotype and quality control thereof
All participants were typed using a custom UKB genotyping chip, and genotypic information for 784256 Single Nucleotide Polymorphic Sites (SNPs) was obtained by genotyping on the autosome in total. Statistical methods of genotype filling were used to fill in large genomic reference panels. The panel consisted of the UK10K haplotype, the thousand genome 3-phase data, and the haplotype reference sample. Approximately nine thousand two million SNPs were co-complemented. As a nano-exclusion criterion, SNPs with a sub-allele frequency <0.1% and a filling precision <0.3 were excluded. After quality control, there were a total of nineteen million SNPs for downstream analysis.
4, genetic Association analysis
Within each gender group, genetic association analysis of the mixed linear model was performed using the BOLT-LMM software. In completing the association score of two sexesAfter analysis, meta analysis was performed on the genetically related signals of both genders using the inverse variance weighted fixed effect model of METAL software. The whole genome significance level was set at α =5 × 10-9. The SNP sites achieving this significance level also meet the gender-specific significance level of α =5 × 10-5. That is, significant correlation signals not only meta-analyzed p-values<5×10-9Simultaneous p-values in both gender groups<5×10-5
The magnitude of the effect of SNPs in the two sex panel was compared using the two-sided z-test. The identified SNP sites were annotated using the Variant Effect Predictor (VEP) software.
5, age Effect
As a typical geriatric disease, the genetic effects of sarcopenia may change with increasing age. Therefore, the patent also evaluates the trend of the SNP effect with age. The specific method is to divide UKB samples into 6 age groups: under age 45 (N =54608), 46-50 (N = 58865), 51-55 (N = 70253), 56-60 (N = 89479), 61-65 (N = 109696), and above age 66 (N = 67342). Genetic association analysis was performed using BOLT-LMM software within each age group. The generated regression coefficients were subjected to meta regression analysis for the average age of each group. The P value of the regression analysis was used as a criterion for the significance.
6, conclusion
SNP site rs33966734 was identified in the UKB sample. Its effect value in the male sample was-0.11, the effect value in the female sample was-0.11 and the overall effect value was-0.11. The effect values did not differ in the male and female groups. The three genotypes AA, CC and AC of rs33966734 correspond to muscle content in the extremities of on average 17.5 kg, 23.3 kg and 20.4 kg, respectively, corresponding to a reduction of about 3 kg for each allele a. Its main genetic association results are shown in Table 1
TABLE 1 genetic association results for rs33966734
Figure RE-GDA0002960827460000041
Note: allele A/C A is the contributing allele.
The site is a missense mutation and is located on chromosome 6CCND3On exon 5 of the gene. The mutation results in the mutation of amino acid 253 of the encoded protein from aspartic acid to glutamic acid. Bioinformatic analysis showed that the mutation had a severe disruptive effect on protein function.
To further determine whether the genetic effect of rs33966734 changes with age, the patent also performs an age-specific analysis. No age-specific effect was found as a result of the analysis.
Taken together, the two allelic sites a and C of the missense mutation rs33966734 are able to distinguish well between high and low muscle groups. When the allele is A, it shows sarcopenia properties, and when it is C, it does not show sarcopenia properties. The allelic form AA results in a decrease in muscle content that ultimately leads to the development of sarcopenia, which is statistically extremely significant and thus can be detected by detecting the site genotype.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Suzhou Jinhao Biotechnology Ltd
<120> a kit for specifically detecting sarcopenia through rs33966734
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 301
<212> DNA
<213> Artificial sequence (Homo sapiens)
<400> 1
ttccccatgt gttgggagct gtccttcccc tgcacctgct gccagatgct atggggggag 60
tgtttgctcc tcactcttct cccatgttcc caggactgcc tgcgggcctg tcaggagcag 120
atcgaagctg cactcaggga nagcctcagg gaagccgctc agaccagctc cagcccagcg 180
cccaaagccc cccggggctc cagcagccaa gggcccagcc agaccagcac tcctacagat 240
gtcacagcca tacacctgta gccctggaga ggccctctgg agtggccact aagcagagga 300
g 301
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Homo sapiens)
<400> 2
ttccccatgt gttgggagct 20
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (Homo sapiens)
<400> 3
ctcctctgct tagtggccac 20
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Homo sapiens)
<400> 4
cacacaggga aagccacagg g 21
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Homo sapiens)
<400> 5
cactcaggga cagcctcagg g 21

Claims (7)

1. A kit for detecting geriatric sarcopenia is characterized in that: the kit comprises a probe and a primer which can specifically detect SNP sites related to senile sarcopenia.
2. The kit of claim 1, comprising a probe and primers capable of specifically detecting rs 33966734.
3. The kit of claim 2, wherein: the kit comprises the nucleotide sequence shown in SEQ ID NO: 2-5.
SEQ ID NO: 2-5 in the preparation of a kit for detecting senile sarcopenia.
Use of rs33966734 as a target for the detection of geriatric sarcopenia.
Use of rs33966734 in the preparation of a kit for the detection of geriatric sarcopenia.
7. A method for detecting geriatric sarcopenia comprising the use of a kit according to any one of claims 1 to 3.
CN202010891138.0A 2020-08-30 2020-08-30 Kit for specifically detecting sarcopenia through rs33966734 Pending CN114107465A (en)

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Publications (1)

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Application publication date: 20220301